Uring HIV-1 fusion with the target cell. The peptides derived from
Uring HIV-1 fusion with the target cell. The peptides derived from the gp41 CHR, e.g. C34 and T1144 are able to bind with viral gp41 N-trimer to block the 6-HB core formation [19,27]. Here, we used a sandwich ELISA and fluorescence native polyacrylamide gel electrophoresis (FNPAGE) to determine if 2DLT, like T1144, possessed inhibitory activity on gp41 6-HB formation in a model system mimicking the gp41 6-HB core formation by mixing the gp41 N36 and C34 (or FAM-labeled C34) peptides at equal molar concentration [17,28]. In the ELISA, 2DLT, like T1144, inhibited the 6-HB formation in a dose-dependent manner with an IC50 of 0.5 ?.06 M, while D1D2 protein at 10 M exhibited no significantinhibition (Figure 4B). Similarly, 2DLT could effectively block 6-HB formation in a dose-dependent manner when it was tested at 5, 10, and 20 M as shown in the FN-PAGE (Figure 4Ca and Cc, lanes 5 to 7), whereas D1D2 protein at the same concentrations showed no significant inhibition (Figure 4Cb and Cd, lines 5 to 7). The D1D2 and 2DLT bands were not observable on the gels because they carry net positive charges, like the N-peptide N36 (lane 1 in Figure 4C) and run in a reversed direction under the native gel condition as previously described [27,29]. These results indicate that 2DLT can interact with the gp41 N-trimer and block the 6-HB core formation between viral gp41 NHR and CHR domains.Lu et PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25447644 al. Retrovirology 2012, 9:104 http://www.retrovirology.com/content/9/1/Page 7 of2DLT could disrupt the function of the CD4-induced gp41 PFI, and caused no significant enhancement of HIV-1 infection in CD4-/CCR5+ GW9662 cost cellsRecently, Haim et al. have demonstrated that binding of sCD4 or CD4-mimetics to HIV-1 gp120 could induce a short-lived PFI N-trimer, resulting in increased bindingAC34-biotin binding ( RLU )a5e+5 4e+5 3e+5 2e+5 1e+5 0 0 10 20 30D1D2 2DLT TInterval between the proteins pulse and addition of C34-biotin at 25 OC ( min )b4e+C34-biotin binding ( RLU ) D1D2 2DLT T3e+2e+1e+0 0 20 40Interval between the proteins pulse and addition of C34-biotin at 4 OC ( min )BActivated infection of HIV-1 (Bal) in the CD4-/CCR5+ cells1600 1400 1200 1000 800 600 400 200 0 1 10 100D1D2 2DLT Tof the C34-Ig protein to the groove on the N-trimer, using a cell-based ELISA as described in Methods. Using a similar approach, we examined whether 2DLT may act on this intermediate to rapidly inactive viruses by the interaction of T1144 domain on it and NHR of gp41 on Env. Indeed, binding of C34 to HIV-1 Env was rapidly enhanced after D1D2 pulse, and then gradually decreased as the time of D1D2 pulse was prolonged at 25 (Figure 5Aa) or 4 (Figure 5Ab), which are coincident with the report by Haim et al. There was no C34 binding immediately after T1144 pulse. Strikingly, the binding ability of C34 to the pulsed Env was lost more rapidly in the 2DLT group than the D1D2 group at 25 (Figure 5Aa) and 4 (Figure 5Ab). These results suggest that because of the presence of T1144 in the bivalent molecule, 2DLT can decay the N-trimer-exposed PFI at a faster rate than D1D2. Therefore, we believe that 2DLT may have a mechanism of action different from D1D2 molecule in inactivating the CD4-induced gp41 PFI. The D1D2 domain in 2DLT may PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 destabilize the gp41 PFI triggered by its binding to gp120, while its T1144 domain may interact with the exposed N-trimer in the viral gp41 to form heterogonous 6-HB, resulting in the stabilization of the gp41 PFI. It has been reported that sCD4 can modestly enhance the.